Tetrodotoxin and manganese ions: effects on electrical activity and tension in taenia coli of guinea pig.

Tetrodotoxin, at concentrations up to 5 x 10(-6) gram per milliliter, has no effect on the spontaneous discharge in the smooth muscle of taenia coli. However, the spontaneous discharge is abolished by Mn(++) at a concentration of 0.5 millimole per liter. The contraction induced by immersing the muscle in isotonic KCl solution is also suppressed in the presence of Mn(++). Because Mn(++) is a specific suppressor of the spike induced by Ca(++) and tetrodotoxin is an inhibitor of the spike induced by Na(+), we suggest that Ca(++) is a charge carrier in the production of spike potential in the smooth muscle and that the entry of intervening Ca(++) through the membrane acts as a trigger for the contraction of smooth muscle.

Myosin II is involved in transmitter release at synapses formed between rat sympathetic neurons in culture.

The presynaptic function of myosin II was studied at cholinergic synapses formed between rat superior cervical ganglion neurons in culture. Immunofluorescent staining showed that myosin II was colocalized with synaptophysin at the presynaptic nerve terminals. Antimyosin II antibody introduced into presynaptic neurons inhibited synaptic transmission. Transmission was also inhibited in a dose-dependent manner by two inhibitors of myosin light chain kinase: a peptide, SM-1, and an organic inhibitor, wortmannin. The inhibition produced by these agents was dependent on presynaptic activity. Extracellularly applied wortmannin also blocked synaptic transmission, but its effects were slower in onset. Wortmannin also decreased postsynaptic potentials and post-tetanic potentiation in intact superior cervical ganglia. These results suggest a model in which myosin light chain kinase phosphorylates myosin, and the resultant change in actin-myosin interactions is involved in neurotransmitter release.

Association of chicken pectoralis muscle phosphorylase with the Z-line and the M-line of myofibrils: comparison with 'amorphin', the amorphous component of the Z-line.

By immunofluorescence technique, glycogen phosphorylase, one of the soluble glycolytic enzymes, was shown to be localized in the Z-line of chicken pectoralis muscle myofibrils, in addition to the M-line, as previously reported by Heizmann, C.W. and Eppenberger, H.M. (Heizmann, C.W. and Eppenberger, H.M. (1978) J. Biol. Chem. 253, 270-277). After extraction of thick filaments by a solution containing pyrophosphate and high salt (Hasselbach-Schneider solution), or after extraction of thin and thick filaments by a solution containing 0.6 M KI, phosphorylase still remained in the Z-line. Amorphin (Mr 85 000) was reported by Chowrashi, P.K. and Pepe, F.A. (Chowrashi, P.K. and Pepe, F.A. (1982) J. Cell Biol. 94, 565-573) as a new Z-line amorphous component. The amino acid composition of amorphin reported by them was very similar to that of phosphorylase b reported by Heizmann and Eppenberger. The partially purified 85 kDa protein, according to Chowrashi and Pepe, showed cross-reactivity to anti-phosphorylase serum and phosphorylase activity. Although amorphin was reported to be eluted from DEAE-column chromatography around the gradient of 0.5 M KCl, little protein was eluted around 0.5 M KCl in our experiments, and the 85 kDa protein which we identified as phosphorylase b was eluted around 0.2 M KCl. Hence, it should be said that the major 85 kDa protein extracted according to Chowrashi and Pepe was phosphorylase b.

A topographical study of the electroplax sodium channel with site-directed antibodies.

Three peptides corresponding to residues (13-23C), (Y1419-1431), and (1809-1820) of the electric eel sodium channel have been synthesized and used to raise antisera in rabbits. All the antibodies produced specifically recognized the corresponding peptides in an ELISA assay. However, their avidities to the channel protein were different. Two antibodies, against the sequence (1809-1820) and (13-23C) which are directed to the C-terminus region and to the adjacent portion of N-terminus, respectively, recognized the 250 kDa channel protein in the immunoblot and an ELISA assay. Binding of the two antibodies to the sodium channel in oriented electroplax membrane vesicles was increased 4-5-fold after permeabilizing the vesicles with 0.01% saponin, implying cytoplasmic orientation for the two peptides. The cytoplasmic orientation of the N- and C-terminal regions were further confirmed by immunogold electron microscopy. By contrast, antibody raised against the sequence (Y1419-1431) which has been proposed to be the transmembrane segment S4 of internal repeat IV, did not react with the channel protein not only after the saponin treatment but also even under the immunoblot conditions following SDS-PAGE. The antibody could recognize fragments of the channel protein after digestion with lysyl endoproteinase, suggesting that the region may apparently form a strictly well-ordered conformation in the transmembrane part of the channel molecule.

Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers.

Chemically skinned fibers from guinea pig taenia caecum were prepared by saponin treatment to study the smooth muscle contractile system in a state as close to the living state as posible. The skinned fibers showed tension development with an increase of Ca2+ in the solution, the threshold tension occurring as 5 X 10(-7) M Ca2+. The maximal tension induced with 10(-4) M Ca2+ was as large and rapid as the potassium-induced contracture in the intact fibers. The slope of the pCa tension curve was less steep than that of skeletal muscle fibers and shifted in the direction of lower pCa with an increase of MgATP. The presence of greater than 1 mM Mg2+ was required for Ca2+-induced contraction in the skinned fibers as well as for the activation of ATPase and superprecipitation in smooth muscle myosin B. Mg2+ above 2 mM caused a slow tension development by itself in the absence of Ca2+. Such a Mg2+-induced tension showed a linear relation to concentrations up to 8 mM in the presence of MgATP. Increase of MgATP concentration revealed a monophasic response without inhibition of Ca2+-induced tension development, unlike the biphasic response in striated muscle. When MgATP was removed from the relaxing solution, the tension developed slowly and slightly, even though the Mg2+ concentrations was fixed at 2 mM. These results suggest a substantial difference in the mode of actin-myosin interaction between smooth and skeletal muscle.

Isolation of the chick myosin alkali light chain gene expressed in embryonic gizzard muscle and transitional expression of the light chain gene family in vivo.

A chick embryonic myosin alkali light chain L23 gene that is expressed transiently at embryonic stages in chick skeletal, cardiac and smooth muscles and in brain continuously from embryo to adult stages, was isolated and characterized. Sequence analysis showed that the exonic sequence of this gene was identical with that of embryonic myosin light chain mRNA except for one base replacement. This gene is a single gene of 5200 bases, which is divided into seven exons by six introns, and the positions of inserts of all the introns are well-conserved as in the skeletal and cardiac muscle myosin alkali light chain genes. Therefore, this embryonic myosin light chain gene can be classified as a member of the myosin alkali light chain gene family, and these three genes may have originated from a common ancestral gene. Transcription of the embryonic light chain gene starts from the same initiation site 33 bases upstream from ATG in embryonic muscle tissues and brain. Comparison of the nucleotide sequence around the promotor region of the embryonic myosin light chain gene with the corresponding regions of the skeletal and cardiac myosin light chain genes showed that the 11-base consensus sequence (TCCTATTTATAG) is present about 100 bases upstream from the transcription initiation site in each gene.

Single chicken cardiac myosin alkali light-chain gene generates two different mRNAs by alternative splicing of a complex exon.

We have isolated and characterized two kinds of cDNA for the chicken cardiac myosin alkali light chain. The sequences of the two cDNAs are identical, except for a notable divergence in part of the 3' untranslated sequence. By analysis of isolated genomic clones, it was shown that the genomic sequences corresponding to the different sequences in the 3' untranslated regions of the two mRNAs were arranged within a limited part of a single stretch of DNA; also the two distinct 3' untranslated regions of the two mRNAs shared part of the last exon, which was 0.6 x 10(3) base-pairs long. There are two canonical acceptor sites available for RNA splicing in the last exon, the first being located at the 5' end of the exon, and the second at 370 base-pairs downstream from this end. Together with analysis by S1 nuclease mapping, the foregoing results lead us to conclude that, by the differential use of these two acceptor sites, a single gene generates two distinct mRNAs of 1.45 x 10(3) base-pairs and 1.1 x 10(3) base-pairs with or without the 5' half of the last exon. The two mRNAs appear to utilize the same modified poly(A) signal, AGTAAA, rather than the authentic AATAAA sequence present about 30 base-pairs downstream from the poly(A) attachment sites. This is probably because another consensus G + T-rich sequence is present at an appropriate distance from the AGTAAA sequence, but not from the AATAAA sequence. The gene for the cardiac myosin alkali light chain has proved to be expressed in ventricular muscle and in atrial and anterior latissimus dorsi muscles, the last of these being characteristic of slow skeletal muscle. In these muscles, two kinds of mRNA for the cardiac myosin alkali light chain, identical with those in ventricular muscle, were expressed and their relative amount in each tissue was almost the same as that in ventricular muscle.

Localization of regenectin in regenerates of American cockroach (Periplaneta americana) legs.

The localization of regenectin, a sucrose-binding C-type lectin, in the regeneration of the cockroach leg was investigated by immunoblotting and immunofluorescence studies. Regenectin was found to appear transiently around developing muscle cells in regenerating legs in the late stage of regeneration. With maturation of the muscles, it disappeared and was not detectable in completely regenerated legs. These findings suggest that regenectin is a cementing substance connecting developing muscle cells. Regenectin was not detected in embryos or nymphal legs at various developmental stages, suggesting that it might not be involved in normal development of embryos and legs.

Increased sarcolemmal permeability in the cerebral artery during chronic spasm: an assessment using DNA-binding dyes and detection of apoptosis.

Alteration of sarcolemmal permeability was evaluated in the cerebral artery after subarachnoid hemorrhage. Significance of membrane dysfunction in the pathogenesis of chronic spasm and contribution of apoptosis were investigated in a canine model. Permeability of the smooth muscle cell (SMC) membrane was assessed by double staining with a hydrophilic (ethidium bromide [EB]) and a lipophilic (Hoechst 33342) DNA-binding dye. Quantitative observations were made with a ultraviolet-fluorescence microscope and a ultraviolet-laser confocal microscope. Occurrence of apoptosis was studied using electrophoresis and TUNEL method. In the normal arteries, nuclei of SMC were stained with Hoechst 33342 but not with EB. In the spastic arteries, SMC in the inner layer of the tunica media were stained with EB. The incidence of EB-positive cells reached maximum on day 7 (45 +/- 19%) and decreased in 2 to 4 weeks (13 +/- 5.2% and 5.0 +/- 2.1%, respectively), in parallel with amelioration of spasm. Electron and light microscopic observations revealed increased density of SMC cytoplasm with widening of the extracellular space. Necrosis was not evident. Apoptosis was not detected by the two methods. These results demonstrate that an augmentation in sarcolemmal permeability takes place during the course of chronic vasospasm and suggest its close correlation to pathogenesis.

Impaired calcium regulation of smooth muscle during chronic vasospasm following subarachnoid hemorrhage.

The intracellular calcium level was determined in the canine basilar artery to investigate whether Ca2+ regulation of its smooth muscle is altered during chronic vasospasm following subarachnoid hemorrhage. A double-hemorrhage model was used. The occurrence of vasospasm was confirmed angiographically 7 days after initial hemorrhage. The intracellular calcium concentration ([Ca2+]i) of smooth muscle was measured using Fura-2. Fluorescence to excitation at 340 and 356 nm was monitored and the ration R340/356 was used as the indicator of [Ca2+]i. When the extracellular calcium concentration ([Ca2+]e) was increased from pCa 8 to 2, [Ca2+]i also increased. In the spastic arteries, the [Ca2+]e - [Ca2+]i curve was elevated as compared with the normal arteries. Treatment with ionomycin elevated the curve in the normal group, but it had little effect in the spastic arteries. Values of [Ca2+]i, calculated in multiples of Kd, were greater in the spastic arteries. Diltiazem (10(-5) mol/L) partially suppressed the augmented [Ca2+]i signal in the spastic arteries, whereas it did not affect the curve in the control group. These results indicate that the calcium regulation of smooth muscle is impaired after subarachnoid hemorrhage, which may contribute to the pathogenesis of chronic vasospasm.

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum.

The amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N-terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium-binding sites (I-VI) has been proposed, two of them (sites II and VI) have lost their calcium-binding function through amino acid replacements, and the other four are able to bind calcium. Six calcium-binding domains are supposed to be derived from two gene duplications of the two ancestral calcium-binding domains. In comparison with the sequence of chick intestinal calcium-binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867-8881], the bovine calcium-binding protein is two amino acid residues shorter at the N-terminus and the other parts show 78.5% identity.

Quantitative analysis of exocytosis visualized by a video-enhanced light/fluorescence microscope reveals two distinct components of exocytosis in RBL-2H3 cells.

Rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca2+-dependent secretion of granules when stimulated with antigen or the Ca2+-ionophore A23187, were observed under a video-enhanced light/fluorescence microscope. Exocytotic events of individual granules were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in the time course. The earlier one was inhibited by selective inhibitors of protein kinase C (Ro31-8425, Ro31-8220, and chelerythrine) and the other was inhibited by an inhibitor of phosphatidate hydrolase, propranolol. Exocytosis by antigen stimulation, however, showed only one peak, which was inhibited by the selective inhibitors of protein kinase C, but not by propranolol. These results indicate that at least two distinct components of exocytosis exist in RBL-2H3 cells.

Distribution of a gelsolin-like 74,000 mol. wt protein in neural and endocrine tissues.

A Ca2(+)-dependent actin binding protein with a molecular weight of 74,000, was purified from bovine adrenal medulla by using deoxyribonuclease I affinity chromatography followed by ion-exchange chromatography and gel filtration. This protein broke actin filaments into fragments and promoted nucleation of actin polymerization in a Ca2(+)-dependent manner as effectively as gelsolin. Proteolytic and immunological comparison with gelsolin which is widely distributed actin-severing protein, indicated that the 74,000 mol. wt protein is a distinct protein, but its domain structure resembles that of gelsolin. Immunoblotting using antibody against this protein showed a tissue-specific distribution. The protein was detected in various endocrine, neuroendocrine and nervous tissues, but not in muscle tissues and plasma which contained relatively large amounts of cytoplasmic and plasma gelsolin. This fact might indicate that this actin-severing protein is involved in the regulation of the secretory process of endocrine and nervous tissues. In the exocytotic process regulated by Ca2+, this protein probably plays a role to free secretory organelles like vesicles from the cytoskeletal network, mainly F-actin, which prevents the movement of secretory vesicles in the resting state.

Differential effects of wortmannin on the release of substance P and amino acids from the isolated spinal cord of the neonatal rat.

1. Effects of wortmannin, an inhibitor of myosin light chain kinase, on the release of substance P and amino acids, GABA and glutamate, were investigated in the isolated spinal cord preparation of the neonatal rat. 2. Wortmannin at 0.5 - 10 microM depressed the release of substance P evoked by high-K+ (90 mM) medium from the spinal cord (IC50 = 1.1 microM). Wortmannin also depressed the high-K+ (70 mM)-evoked release of substance P from cultured dorsal root ganglion neurons of neonatal rats. In contrast, the high-K+ (90 mM)-evoked release of GABA and glutamate from the spinal cord was not affected by wortmannin (0.1 - 10 microM). 3. Upon stimulation of a dorsal root, a monosynaptic reflex and a subsequent slow ventral root depolarization were evoked in the ipsilateral ventral root of the same segment in the isolated spinal cord preparation. The magnitude of the slow ventral root depolarization was depressed gradually to about 70% of the control during the course of 30 min under wortmannin (1 microM). In contrast, the monosynaptic reflex was unaffected by wortmannin. 4. Immunofluorescent staining revealed that immunoreactivities of substance P and myosin II were colocalized at presynaptic terminals in the dorsal horn of the neonatal rat spinal cord. 5. The present results suggest that myosin phosphorylation by myosin light chain kinase may play a crucial role in the release of substance P, but not in the release of GABA and glutamate in the neonatal rat spinal cord. This may reflect a difference in the exocytic mechanisms of substance P-containing large dense core vesicles and amino acid-containing small clear vesicles.